Synergistic combination therapy delivered via layer‐by‐layer nanoparticles induces solid tumor regression of ovarian cancer

Abstract The majority of patients with high grade serous ovarian cancer (HGSOC) develop recurrent disease and chemotherapy resistance. To identify drug combinations that would be effective in treatment of chemotherapy resistant disease, we examined the efficacy of drug combinations that target the three antiapoptotic proteins most commonly expressed in HGSOC—BCL2, BCL‐XL, and MCL1. Co‐inhibition of BCL2 and BCL‐XL (ABT‐263) with inhibition of MCL1 (S63845) induces potent synergistic cytotoxicity in multiple HGSOC models. Since this drug combination is predicted to be toxic to patients due to the known clinical morbidities of each drug, we developed layer‐by‐layer nanoparticles (LbL NPs) that co‐encapsulate these inhibitors in order to target HGSOC tumor cells and reduce systemic toxicities. We show that the LbL NPs can be designed to have high association with specific ovarian tumor cell types targeted in these studies, thus enabling a more selective uptake when delivered via intraperitoneal injection. Treatment with these LbL NPs displayed better potency than free drugs in vitro and resulted in near‐complete elimination of solid tumor metastases of ovarian cancer xenografts. Thus, these results support the exploration of LbL NPs as a strategy to deliver potent drug combinations to recurrent HGSOC. While these findings are described for co‐encapsulation of a BCL2/XL and a MCL1 inhibitor, the modular nature of LbL assembly provides flexibility in the range of therapies that can be incorporated, making LbL NPs an adaptable vehicle for delivery of additional combinations of pathway inhibitors and other oncology drugs.


| INTRODUCTION
Ovarian cancer remains the leading cause of death among gynecologic malignancies in the United States, with an overall 5-year survival rate of 41%-49% that drops to 24%-30% for those with late stage diagnoses (Stage III/IV). 1,2 First line treatment of high grade serous ovarian cancer (HGSOC), the most common form of ovarian cancer, consists of surgical cytoreduction and platinum-based chemotherapy. This often fails to eradicate residual disease, resulting in 70% patient relapse within 5 years. 3 What drives relapse-particularly in patients with advanced-stage solid ovarian cancers-is cancer cell resistance to nonspecific, cytotoxic platinum-based treatment. 4 In contrast to traditional chemotherapies, targeted therapies inhibit specific oncogenic pathways essential for tumor cell survival.
For HGSOC, which lacks many identifiable therapeutically treatable point mutations, targeting the oncogenic and often-dysregulated mitochondrial apoptotic pathway is especially attractive. 5 BCL-2 homology domain 3 (BH3) mimetics are drugs that mimic the inhibitory actions of proapoptotic BH3 only proteins by binding to antiapoptotic BCL-2 family proteins (i.e., BCL2, BCL-XL, MCL1), leading to reactivation of apoptotic machinery. 6,7 Inhibiting the action of antiapoptotic BCL-2 family proteins has already shown promise in treating a number of human hematopoietic malignancies 8 and non-ovarian solid tumor xenografts. 6,9,10 Although studies have reported synergistic interactions from co-inhibition of BCL-XL and MCL1 antiapoptotic proteins, [11][12][13][14][15][16][17][18][19][20] this inhibitor combination had yet to be investigated in treatment of solid tumors of HGSOC. Clinical evaluation of HGSOC tumors has correlated high expression of BCL-XL/MCL1 with increased chemoresistance/recurrence. These two proteins were also found to be commonly expressed in HGSOC patient derived xenograft (PDX) models, 21,22 suggesting that targeted inhibition of BCL-XL and MCL1 proteins is a promising strategy for overcoming mechanistic resistance.
Compared to other BH3 mimetics, 8,23 the translation of small molecule BCL-XL and MCL1 targeted therapies is less clinically advanced due to toxicities and pharmacological challenges. While targeted inhibitors are selective for specific oncogenic pathways, their delivery to cells is nonspecific. Both BCL-XL and MCL1 are present in healthy adult, non-malignant tissue, and studies have reported thrombocytopenia 24 and cardiac toxicities 2526 for some BCL-XL and MCL1 inhibitors. Additionally, many BCL-XL and MCL1 inhibitors exhibit poor solubility and unfavorable pharmacokinetics, and thus represent suitable drug candidates that would benefit from the use of a delivery vehicle. 6 Layer-by-layer nanoparticles (LbL NPs), formed from the electrostatic assembly of oppositely charged polyelectrolytes, have been shown to extend circulation time, limit off-target toxicities, and enhance targeted therapeutic delivery to cancer cells. [27][28][29][30][31] We have recently shown that LbL NPs designed with appropriate outer layer chemistries exhibit greater than 80% ovarian cancer tumor accumulation when delivered intraperitoneally and can be further functionalized with outer layer targeting chemistry that enhances penetration of solid tumors. [32][33][34] Modular LbL architecture can be adapted for encapsulation of a wide range of therapeutics [27][28][29][30][31]34,35 and cancer cell targeting chemistries, 32,33 and has scalable synthesis. 36 We developed a synergistic LbL NP combination therapy for targeted delivery and treatment of HGSOC. We demonstrate that dual inhibition of BCL2/XL and MCL1 is synergistic in 4 out of 5 tested HGSOC models, and that treatment efficacy is significantly enhanced by LbL NP-mediated delivery to cancer cells in vitro. In a metastatic mouse model of ovarian cancer, we observed that the LbL NP combination therapy eliminated nearly all metastatic solid tumor lesions at lower total drug dosages (6 mg/kg) than what has been previously reported in the literature for other mouse models. 10,19,37 Additionally, we determined no overt toxicities from LbL NP combination treatment. Moreover, we demonstrate in vivo that it is the combination of BCL2/XL and MCL1 inhibition, rather than singular inhibition of either BCL2/XL or MCL1 that successfully induces regression of metastatic solid tumor lesions in HGSOC. We report here a synergistic combination therapy, delivered via LbL NPs, as a potential treatment for overcoming platinum resistance and inducing significant regression of solid tumors in HGSOC.  were synergistic in most tested HGSOC cells, degree of synergy varied by PDX, and the effect was additive in DF20. Tumor cell heterogeneity is a potential cause of the observed differences in synergistic activity. 41,42 Previous work has shown that the relative abundance of antiapoptotic proteins varies between PDX models. 43 Encapsulating inhibitors against BCL2, BCL-XL, and MCL1 enables pathway inhibition across multiple models.

| Loading and characterization of LbL NPs
LbL NPs were formulated with a drug-loaded core and tumortargeting chemistry (Figure 2a) to improve the delivery of the lipophilic BCL2/XL and MCL1 inhibitors into cancer cells (Figure 2b).
When used in free form with patients, these drugs are typically delivered systemically in oral formulations which cannot be targeted to the tumor. We elected to use poly-lactic-co-glycolic acid (PLGA) to encapsulate the small molecule inhibitors for its biocompatibility, biodegradability, and anionic surface charge compatible with charge-based LbL deposition. Drug cores were then layered with cationic poly-L-arginine (PLR), which has been previously used to promote endosomal escape of LbL NPs into the cytoplasm. 29,35,44,45 NPs were terminally layered with tumor-targeting hyaluronic acid (HA) or poly-L-aspartic acid (PLD) outer layer chemistry. We selected HA because it is a known ligand of the CD44 receptor, overexpressed on HGSOC cells, 46 and has been previously used by our lab and several others to target the CD44 receptor. [27][28][29][30]32 We selected PLD based on our recent findings that LbL NPs coated with a surface layer of PLD have selective targeting interactions with a HGSOC model (OVCAR8). 32 LbL NPs were formulated as single-drug NPs (BCL2/XLi NP, MCL1i NP) and combination NPs (Combo-NP, co-encapsulation of BCL2/XLi and MCL1i) (Figure 2b). The method of nanoprecipitation 47,48 was utilized to load the inhibitors into the PLGA core. Tangential flow filtration 36 was employed to remove unencapsulated drug prior to LbL deposition. High performance liquid chromatography (HPLC) was used to determine the encapsulation efficiency (EE) in the resulting LbL NPs ( Figure 2b). We observed slightly higher EE for BCL2/XLi (Log P = 6.593) than MCL1i (Log P = 3.921). For Combo-NPs, wt% loading of BCL2/xLi (18.6 ± 3.78) is slightly greater than wt% loading of MCL1i (12.8 ± 1.48). Accounting for differences in molecular weight, Combo-NPs are loaded in approximately a 55:45 molar ratio of BCL2/ xLi to MCL1i.

| LbL NPs target HGSOC models in vivo
LbL NPs with different outer layers were assessed for in vivo colocalization with HGSOC models to select for optimal LbL NP targeting chemistry. OVCAR8-Nude and DF09-NSG models have significant tumor lesions in the omentum and along the fat covering the upper genital tract. Due to this disseminated tumor formation, colocalization of tumor bioluminescence signal and LbL NP fluorescence signal provides a stronger and more nuanced evidence of LbL NP homing to tumor cells. Colocalization analysis also allowed comparison of NP targeting across different tumor models more effectively; while the OVCAR8-Nude model forms tumor nodules, the DF09-NSG model forms a thin layer of tumor cells caking surfaces of the peritoneal space. Differences in tumor formation between models make NP accumulation in solid tumor impossible to compare across models using a "total tissue accumulation" approach.
Cy7-labeled HA-and PLD-coated LbL NPs (without inhibitors) were formulated to track NP localization in vivo and have similar size and charge to drug-loaded HA-and PLD-coated LbL NPs ( Figure S1).    Figure S2). The differences in degree of NP-tumor colocalization suggest that the optimal targeting chemistry of LbL NPs may depend on differences in cell surface protein expression among tumor models. 40 The LbL architecture provides a tunable platform that enables altering of the outer layer targeting chemistry toward given tumor cell types, independent of drug composition. It is anticipated that different tumor cell surface markers or overexpressed proteins might be targeted with ligands or surface chemistries. The HA outer layer homes most efficiently to OVCAR8 due to its characteristic CD44 overexpression which is also found in some other high grade ovarian cancer models. 50,51 Our lab has demonstrated high affinity of the PLD outer layer for several ovarian cancer cell lines 32 ; we have proposed that these less-specific interactions may involve hydrogen bond  Correlation is higher for PLD-NPs (0.24 ± 0.15) is higher than HA-NPs (0.01 ± 0.04). Values of R are given as mean ± standard error. ImageJ Coloc2 plugin was used to calculate correlation coefficients. To prevent bias from background signal and ensure that correlation coefficients are only being calculated from tissue signal, regions of interest (ROIs) of the organs were generated prior to running the analysis interactions between PLD and glycans expressed on a number of HGSOC cancer cell types. In vivo imaging system (IVIS) images depicting relative levels of NP accumulation in tumor and healthy tissue show that NP accumulation was highest in tumors covering the omentum/UGT, and in the liver and kidney. NP accumulation was lowest in the heart, spleen, and lungs ( Figure S3).

| LbL NP-mediated delivery improves in vitro efficacy of combination treatment
To assess the efficacy of LbL NP-mediated delivery of BCL2/XLi and    (Figure 5a). All doses were administered IP at the MFD.
A full panel of toxicological metrics was used to assess drugrelated toxicities (Figure 5b). Mice were monitored throughout treatment for signs of distress and significant change in body weight.
Exactly 24 h after the last dose was administered, blood was collected and assayed for platelet, erythrocyte, and leukocyte health (via complete blood counts [CBCs]) and liver enzyme/protein levels via serum chemistry. Tissue sections were stained with hematoxylin and eosin to look for inflammation and major changes to tissue morphology.
NCr Nude mice experienced no significant weight loss (Figure 5c).
Platelet levels were within the normal ranges provided by MIT's Division of Comparative Medicine Comparative Pathology Lab (DCM CPL) (Figure 5d). While we observed that liver enzyme levels spiked in the 1D group after a single dose, these levels were within the normal range for 7D and 14D groups (Figure 5e). To assess hepatic function, protein levels were analyzed, and we found no lasting toxicity from continuous dosing treatment (Figure 5f). Lasting hepatic damage impacting liver function is expected to cause an increase in total bilirubin, and a decrease in albumin and total protein levels. All treatment groups were within normal ranges for total bilirubin, albumin, and protein levels. Tissue sections reveal no inflammation or major morphological changes to the liver (Figure 5g). Combo-NP treatment did not cause damage to other major organs (kidney, spleen, heart, lungs) and levels of erythrocytes and leukocytes were within normal ranges ( Figures S7 and S8). We also observed that NSG mice continually lost weight throughout   proteins. 17 All formulations tested in vivo on OVCAR8 NCr Nude tumorbearing mice were well tolerated; mice did not experience significant weight loss ( Figure S9C). Levels of liver enzymes, protein levels, and blood urea nitrogen (BUN)/creatine were not statistically different among treatment groups suggesting normal liver and kidney function ( Figure S10A,B). Mice did not experience a reduction in platelets with treatment ( Figure S10C). And erthryocyte/leukocyte counts were not statistically different among treatments ( Figure S11). Thus, all LbL NP treatments are well tolerated and demonstrably safe.  mitochondrial apoptosis pathway increases efficacy over free drug. 56  However, they face a number of challenges in their clinical translation.

| DISCUSSION
LbL NP encapsulation and targeted delivery can be used to safely and effectively deliver such synergistic combination therapies. We devel-

ACKNOWLEDGMENTS
We would like to thank the KI Histology core, in particular Kathy

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.